Cleft palate represents one of the most common congenital birth defects in the human population. Despite recent advancements in medical intervention, babies born with cleft palate often suffer multiple handicaps that significantly compromise the quality of their lives. The causes of cleft palate are numerous, including multiple genetic and environmental factors (Chai and Maxson, 2006; Dixon et al., 2011). Recent studies have identified a large number of signaling pathways that regulate hard palate development. However, the regulatory mechanisms for development of the soft palate are largely unknown. The soft palate in humans consists of five muscles that are functionally highly active in our daily lives as they form a critical component of our oropharyngeal complex. Cleft of the soft palate leads to misorientation of these muscles, causing oropharyngeal deficiency and adversely affecting speech, swallowing, breathing and hearing. Our preliminary studies have shown that TGF-? signaling is specifically required in cranial neural crest-derived cells (CNCCs) for regulating tissue-tissue interactions to control soft palate development. Furthermore, we have uncovered important TGF-? downstream target genes, such as Dlx5 and Runx2, which may play crucial roles in regulating soft palate development. Our preliminary data show that Runx2 is specifically required for regulating CNCC and myogenic progenitor interaction during soft palate development. Loss of Runx2 in CNCCs leads to cleft of the soft palate. Based on this foundation, we will test the hypothesis that TGF-? regulates the expression of key transcription factors, such as Dlx5 and Runx2, which function to mediate CNC and myogenic cell interaction to control the patterning and development of muscles in the soft palate. Our Specific Aims are: (1) To investigate how Alk5-mediated TGF-? signaling in CNCCs controls muscle patterning and development through tissue-tissue interactions. We will analyze the molecular and cellular defects in the soft palate of Alk5 mutant mice. We will test how TGF-??downstream signaling molecules play key roles in regulating cell-cell interactions using an organ-on-a-chip model and explore the potential to rescue the myogenic defect in the soft palate of Alk5 mutant mice. (2) To investigate the functional significance of Dlx5 signaling in CNCCs in regulating the fate of myogenic progenitors during soft palate muscle development. We will test how Wnt signaling and chromatin remodeler Arid1a, acting downstream of Dlx5, may play a key role in mediating tissue- tissue interaction to control soft palate muscle development. (3) To investigate how Runx2, acting downstream of Alk5-mediated TGF-? signaling, may exert its regulatory role in controlling tissue-tissue interactions to regulate soft palate development. We will also test whether Dlx5/Runx2 interaction may play a key role in regulating soft palate development.